Isolation of polymerization-competent vimentin from porcine eye lens tissue

Abstract

Most higher cells contain ‘10 nm filaments’ (intermediate filaments) as third cytoskeletal component in addition to microtubules and actincontaining microfilaments. Immunological and gelelectrophoretic studies have distinguished several distinct intermediate filament subclasses, each related to certain cell and tissue types (review [ 1,2]). Whereas neurofilaments, glial filaments, muscle desmin filaments and epidermal tonofilaments are readily available from corresponding tissues, it has consistently remained difficult to obtain vimentin in larger amounts. Vimentin is found in mesenchymal cells and also expressed in many, if not all, permanent cell lines [ 1,4]. Denatured vimentin has been purified in small amounts by extraction of cytoskeletons of cultured cells using preparative gel electrophoresis [l-4]. In addition,native intermediate filaments have been isolated in bundled form from cultured BHK 21 cells [5], but subsequent studies revealed that they contain not only vimentin, but also desmin [6,7] and some higher molecular weight (or M,) proteins [8]. In order to explore the molecular basis of different intermediate filaments, protein-chemical characterization is required and in the case of vimentin an easy purification scheme is demanded. Here we report that eye lens tissue, which is known to contain vimentin by immunological criteria [9] is an excellent source to obtain purified vimentin in good yield. The isolated protein renatures spontaneously into filaments after urea treatment. The purification scheme described eliminates the need for costly tissue culture facilities and provides amounts suitable for further biochemical and protein-chemical characterizations.